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Effects of exogenous cysteine on inorganic and organic arsenicals-induced cytolethality.

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APPLIED ORGANOMETALLIC CHEMISTRY
Appl. Organometal. Chem. 2006; 20: 549–556
Published online 15 May 2006 in Wiley InterScience
(www.interscience.wiley.com) DOI:10.1002/aoc.1072
Speciation Analysis and Environment
Effects of exogenous cysteine on inorganic and organic
arsenicals-induced cytolethality†
Teruaki Sakurai1 *, Chikara Kojima1 , Michael P. Waalkes2 and Seiichiro Himeno1
1
Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho,
Tokushima 770-8514, Japan
2
Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of
Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA
Received 20 December 2005; Accepted 2 March 2006
Inorganic arsenicals are distinctly toxic and carcinogenic to humans. Inorganic arsenite (AsIII )
and arsenate (AsV ) are enzymatically methylated to monomethylarsonic acid (MMAsV ) and
dimethylarsinic acid (DMAsV ) in mammals. Recent reports indicate that cytotoxic trivalent methylated
arsenicals are produced through methylation of inorganic arsenicals and are involved in arsenic
poisoning. Some previous studies have suggested that a typical thiol reagent cysteine (Cys) can
reduce pentavalent arsenicals to trivalent arsenicals and might be able to enhance arsenic cytolethality.
However, not much is known concerning the effects of exogenous Cys on the cytolethality of arsenicals.
In this study, we examined the effects of exogenous Cys on the cytolethality induced by inorganic
and organic arsenicals using rat liver cells. Cys prevented inorganic AsIII -induced cytolethality. In
contrast, when more than 5 mM Cys was incubated with millimolar levels of MMAsV or DMAsV ,
MMAsV - and DMAsV -induced cytolethality significantly increased; this cytolethality might have
been caused by the generation of trivalent methylated arsenicals. However, Cys at a concentration of
less than 5 mM decreased the MMAsV - and DMAsV -induced cytolethality. These findings suggest that
high concentrations of both arsenicals and Cys are required to form trivalent methylated arsenicals
and to induce significant cytolethality. Copyright  2006 John Wiley & Sons, Ltd.
KEYWORDS: arsenic; cysteine; dimethylarsinic; monomethylarsonic; Cys; Cys conjugate
INTRODUCTION
Arsenic is a metalloid element that is widely distributed
in the environment as inorganic trivalent (arsenite; AsIII )
or pentavalent (arsenate; AsV ) forms,1 and its toxicity has
been known since ancient times. In Asia and the Americas,
chronic arsenic poisoning has occurred as a result of the
consumption of water from wells drilled into arsenic-rich
strata. Epidemiological studies have provided clear evidence
that inorganic arsenicals are human carcinogens with target
†
This paper is based on work presented at the 12th Symposium
of the Japanese Arsenic Scientists’ Society (JASS) held 5–6
November 2005 in Takizawa, Iwate Prefecture, Japan.
*Correspondence to: Teruaki Sakurai, Laboratory of Molecular
Nutrition and Toxicology, Faculty of Pharmaceutical Sciences,
Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514,
Japan.
E-mail: teruaki@ph.bunri-u.ac.jp
Copyright  2006 John Wiley & Sons, Ltd.
sites including liver, skin, lung, kidney and urinary bladder.2
On the other hand, inorganic AsIII has emerged as a potent
chemotherapeutic agent with remarkable efficacy for certain
human cancers such as acute promyelocytic leukemia.3,4
It would thus appear that environmental and iatrogenic
exposure to arsenicals will continue to be common.
In humans and numerous experimental animals, inorganic
arsenicals are rapidly absorbed from the gastrointestinal
tract and reduced to AsIII .5,6 Subsequently, it may be
enzymatically methylated in the liver or other organs
into organic arsenicals such as monomethylarsonic acid
(MMAsV ) and dimethylarsinic acid (DMAsV ).7 MMAsV
and DMAsV are the major organic pentavalent arsenic
metabolites in human urine after exposure to inorganic
arsenicals.5,7 It is believed that methylation of inorganic
arsenicals results in a reduction in general toxicity, as
indicated by their increased in vivo lethal dose in 50% of a
population (LD50 ) and in vitro lethal concentration in 50% of a
550
T. Sakurai et al.
population (LC50 ).8,9 However, the role of the methylation
of inorganic arsenicals is not entirely clear and recent
studies have increasingly suggested that the methylation
of inorganic arsenicals is not a universal detoxification
mechanism. It has been reported that trivalent methylated
arsenicals such as monomethylarsonous acid (MMAsIII )
and dimethylarsinous acid (DMAsIII ) are found in urine
collected from individuals who have been exposed to high
concentrations of inorganic arsenicals;10,11 further, synthetic
trivalent methylated arsenicals such as monomethylarsine
oxide (MMAsIII O) and iododimethylarsine (DMAsIII I) were
more cytotoxic in vitro than inorganic and pentavalent
methylated arsenicals.12 It is generally believed that arsenicals
react with various thiol reagents, and a typical thiol
reagent, cysteine (Cys), might reduce pentavalent methylated
arsenicals to trivalent methylated arsenicals in human body.
Some studies have reported that Cys enhance arsenic
cytolethality,13 – 15 however, not much is known concerning
the effects of exogenous Cys on the cytolethality of arsenicals.
In the present study, we observed the true effects of
exogenous Cys on inorganic and organic arsenicals-induced
cytolethality. This study may provide important information
on the reaction between various arsenicals and thiol reagents.
EXPERIMENTAL
Chemicals
Sodium arsenite (AsIII ), sodium arsenate (AsV ), and DMAsV
were purchased from Wako Pure Chemical Co. (Osaka,
Japan). MMAsV was purchased from Trichemical Co.
(Yamanashi, Japan). These arsenicals were recrystallized
twice, and their purities were >99.9% as determined by gas
chromatography–mass spectrometry.8,9 Endotoxin contamination of these arsenicals was not detected (<0.0000003%,
wt/wt) using the endotoxin-specific limulus test (Seikagaku
Co., Tokyo, Japan). L-Buthionin-(S,R)-sulfoximine [BSO; an
inhibitor of γ -glutamylcysteine synthetase which decreases
cellular reduced glutathione (GSH) levels], L-cysteine (Cys)
and N-acetyl-L-cysteine (NAC) were purchased from Sigma
Chemical Co. (St Louis, MO, USA). L(−)-Cystine (Cys–Cys)
was purchased from Wako Pure Chemical Co. (Osaka, Japan).
Cell culture
The TRL 1215 cells are nontumorigenic adhesive rat epithelial
liver cells originally derived from the liver of 10-day old
Fisher F344 rats.16 TRL 1215 cells were cultured in William’s
E medium (Sigma) supplemented with 10% heat-inactivated
fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin G
and 100 µg/ml streptomycin under a humidified atmosphere
of 5% CO2 :95% air at 37 ◦ C.
Speciation Analysis and Environment
plated on flat-bottomed 96-well tissue culture plates and
allowed to adhere to the plate for 24 h at 37 ◦ C, at which time
the medium was removed and replaced with 100 µl/well
fresh medium containing the various test samples. Cells
were then incubated with test samples for an additional
48 h at 37 ◦ C. After incubation, cells were washed twice
with warmed phosphate-buffered saline (pH 7.4) to remove
non-adherent dead cells, and cell viability was determined
by the AlamarBlue assay, which is similar to microtitertetrazolium (MTT) assay and measures metabolic integrity.8,9
Briefly, after incubations with test samples and replacement
with 100 µl/well fresh media, 10 µl/well AlamarBlue solution
(Iwaki Grass Co., Chiba, Japan) was added directly to the 96well plates, incubated for 4 h at 37 ◦ C, and the absorbance at
570 nm (referenced to 600 nm) was measured by a microplate
reader model 550 (Bio-Rad Laboratories, Hercules, CA, USA).
Data were expressed as metabolic integrity using the values
from control cells, which were incubated with medium alone
for 48 h at 37 ◦ C, as 100%.
Thin layer chromatography (TLC)
TLC was performed with high performance TLC (HPTLC)
plates silica gel 60 F 254 (Merck, Darmstadt, Germany)
with a developing solvent of ethyl acetate : acetic acid : water
(3 : 2 : 1). Iodide vapor was used for the detection of the various
arsenicals.8,17
MTS assay
MTS assay was performed using Cell Titer 96 NonRadioactive Cell Proliferation Assay Kit (Promega Co., Madison, WI).18,19 This assay measures the amount of formazan
produced by the metabolic conversion of Owen’s reagent [3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] by Cys. The
MTS is the subject of U.S. patent no. 5185450 and is licensed to
Promega Co. Briefly, 15 µl of various concentrations of arsenicals were incubated with 15 µl of 1 mM Cys, 20 µl the kit’s MTS
solution, and 100 µl phosphate-buffered saline (pH 7.4) in 96well microtiter plate at 37 ◦ C during 120 min. The absorbance
at 490 nm of formazan was measured by a microplate reader.
Statistical analysis
The data represent the mean plus or minus the standard
error of the mean (SEM) and statistical evaluations were
performed by Student’s t-test or one-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test as
appropriate.20 A value of p < 0.05 was considered significant
in all cases.
RESULTS
Assay for cytolethality
Effect of exogenous L-cysteine (Cys) on the
cytolethality of AsIII , AsV , MMAsV or DMAsV
Cells were isolated by trypsinization, washed twice and
resuspended in fresh medium. 2 × 104 cells/100 µl/well were
The effect of exogenous Cys on the cytolethality of AsIII ,
AsV , MMAsV or DMAsV in vitro was examined. TRL 1215
Copyright  2006 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
Speciation Analysis and Environment
Effects of cysteine on arsenicals-induced cytolethality
Table 1. Effect of exogenous Cys on arsenicals-induced cytolethality
Cys
BSO
Medium
AsIII
AsV
MMAsV
DMAsV
10 µM
25 µM
25 µM
500 µM
2.5 mM
10 mM
5 mM
10 mM
−
−
+
−
−
+
+
+
100.0 ± 6.5
83.8 ± 4.5
8.5 ± 0.6
99.0 ± 1.8
15.8 ± 1.7
93.8 ± 1.0
34.1 ± 0.2
50.8 ± 2.5
6.0 ± 1.8
80.4 ± 3.6
82.7 ± 0.6
63.6 ± 2.7a
97.8 ± 1.1
14.0 ± 0.7
94.4 ± 2.1
31.1 ± 2.5
75.3 ± 1.3b
0.5 ± 0.5
103.3 ± 5.5
12.7 ± 4.3a
0.0 ± 0.0a
3.2 ± 0.6a
3.6 ± 1.9
94.6 ± 5.6
0.7 ± 0.0a
97.3 ± 2.9a
78.8 ± 2.7a
80.8 ± 1.3
15.5 ± 7.8a
0.0 ± 0.0a
0.0 ± 0.0a
4.7 ± 3.4
48.5 ± 5.5a,c
0.0 ± 0.0a
93.9 ± 1.3a
0.4 ± 0.4c
TRL 1215 cells were preincubated with or without 50 µM BSO for 24 h. These cells were further exposed to AsIII (10 or 25 µM), AsV (25 or 500 µM),
MMAsV (2.5 or 10 mM), or DMAsV (5 or 10 mM) with or without 10 mM Cys in the presence or absence of 50 µM BSO for 48 h. Cellular viability was
then assessed by the AlamarBlue assay. Results were expressed as metabolic integrity using the values from control cells, which were incubated
with medium alone for 48 h at 37 ◦ C, as 100%. Arithmetic mean plus/minus SEM of three separate experiments performed in triplicate, n = 9.
a p < 0.001, in comparison to the cells exposed to same concentrations of AsIII , AsV , MMAsV , or DMAsV alone,
b p < 0.01, c p < 0.001, in comparison to the cells simultaneously treated to both AsIII , AsV , MMAsV or DMAsV and BSO.
cells were preincubated with or without 50 µM BSO for 24 h.
These cells were subsequently exposed to AsIII , AsV , MMAsV
or DMAsV with or without 10 mM Cys in the presence or
absence of BSO for 48 h, and then the cellular viability was
assessed. We previously reported that AsIII , AsV and DMAsV
showed significant cytolethality in TRL 1215 cells; their LC50
values after 48 h exposure were 20 µM, 150 µM and 4.8 mM,
respectively, and that MMAsV was not cytotoxic even at
concentrations over 5 mM.8,17 – 19,21 – 23 Exogenous Cys alone
showed weak cytolethality in TRL 1215 cells at over 5 mM;
the LC50 for Cys after 48 h exposure was 12.1 or 12.5 mM in
the presence or absence of BSO pretreatment, respectively.
Exogenous Cys did not augment cellular GSH levels in TRL
1215 cells during this incubation period (data not shown).
BSO (50 µM) alone did not influence cell viability.
As shown in Table 1, exogenous Cys at a concentration of
10 mM significantly decreased the cytolethality of 25 µM AsIII ,
although a decrease in the cytolethality was not observed in
cellular GSH-depleted cells. The cytolethality of AsV was not
affected by 10 mM Cys. The addition of 10 mM Cys strongly
enhanced the cytolethality of 2.5 mM MMAsV in cellular
GSH-depleted cells. The cytolethality of 5 mM DMAsV was
significantly decreased by the addition of 10 mM Cys in the
absence of 50 µM BSO. The addition of 10 mM Cys enhanced
the cytolethality of 10 mM DMAsV in cellular GSH-depleted
cells.
TRL 1215 cells were preincubated with or without 50 µM
BSO for 24 h, and these cells were further exposed to 10 mM
MMAsV or DMAsV with or without various concentrations
of Cys in the presence or absence of 50 µM BSO for 48 h.
The addition of <1.25 mM Cys did not affect MMAsV induced cytolethality; however, the addition of ≥1.25 mM
Cys strongly prevented MMAsV -induced cytolethality in the
absence of BSO [Fig. 1(A)]. The addition of 1.25–5 mM Cys
prevented DMAsV -induced cytolethality in the absence of
Copyright  2006 John Wiley & Sons, Ltd.
BSO; however, the addition of ≥5 mM Cys enhanced the
cytolethality in cellular GSH-depleted cells [Fig. 1(B)].
The same results were obtained when the cells were
simultaneously exposed to both arsenicals and N-acetyl-Lcysteine (NAC), which is similar in structure to Cys (data not
shown).
Chemical reactions of AsIII , AsV , MMAsV and
DMAsV with Cys in vitro
We determined the plausibility of chemical reactions
occurring between arsenicals and Cys. For this, we used
MTS assays that indirectly demonstrated such reactions by
measuring the competitive inhibitory effects of arsenicals
on the reaction between Cys and MTS.18,19 As shown in
Fig. 2, while AsIII strongly enhanced the Cys–MTS reaction
in a dose-dependent manner, AsV enhanced this reaction to
a slight extent. However, MMAsV and DMAsV significantly
inhibited the Cys–MTS reaction in a dose-dependent manner.
MTS did not react with any of the arsenicals or Cys–Cys alone
(data not shown).
MMAsV and DMAsV easily combine with Cys
in water
In order to determine the likelihood of the MMAs–Cys
conjugate production, 1 mM MMAsV was incubated with
or without 1, 4, 5 or 10 mM Cys in distilled water for 1 h
at 37 ◦ C. After the incubation, these mixtures were applied
to an HPTLC plate and separated using ethyl acetate : acetic
acid : water (3 : 2 : 1). Separated compounds were detected
with iodide vapor. The TLC method have been authorized
for separating and detecting of arsenic compounds.8,17,24 As
shown in Fig. 3(A), Cys [lane 1; relative mobility (Rf ) = 0.45]
and Cys–Cys (lane 2; Rf = 0.11) spots were detected with
iodide vapor, but MMAsV was not detected under these
experimental conditions (lane 3). The Cys spot did not appear
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
551
552
Speciation Analysis and Environment
T. Sakurai et al.
Figure 1. Effect of exogenous Cys on the cytolethality of
MMAsV or DMAsV . TRL 1215 cells were preincubated with
(×) or without ( ) 50 µM BSO for 24 h at 37 ◦ C. After the
preincubation, these cells were exposed to 10 mM MMAsV (A) or
DMAsV (B) with or without various concentrations of Cys in the
presence (×) or absence ( ) of 50 µM BSO for 48 h at 37 ◦ C.
Cellular viability was then assessed by the AlamarBlue assay.
Results are expressed as metabolic integrity using the values
from control cells, which were incubated with medium alone
for 48 h at 37 ◦ C, as 100%. Arithmetic mean plus/minus SEM
of three separate experiments performed in triplicate, n = 9.
a
p < 0.001, in comparison to the cells exposed to MMAsV or
DMAsV alone, b p < 0.01; c p < 0.01, in comparison to the cells
simultaneously treated to both MMAsV or DMAsV and BSO,
d
p < 0.001.
°
°
when 1 mM Cys was incubated with 1 mM MMAsV (lane 4),
and a putative MMAs–Cys conjugate spot was detected at a
different position from the Cys and Cys–Cys spots (lanes 5–7;
Rf = 0.18) after incubating 1 mM MMAsV with ≥4 mM Cys.
We also examined the production of the DMAs–Cys
conjugate from DMAsV and Cys using the same HPTLC
system; 1 mM DMAsV was incubated with or without 1,
3, 5 or 10 mM Cys in distilled water for 1 h at 37 ◦ C, and
these mixtures were then applied to an HPTLC plate and
separated. As shown in Fig. 3(B), DMAsV was not detected
under these experimental conditions (lane 3). When 1 mM Cys
was incubated with 1 mM DMAsV , the Cys spot disappeared
Copyright  2006 John Wiley & Sons, Ltd.
Figure 2. Inhibitory effect of arsenicals on the Cys-MTS
reaction in vitro. (A) Cys was incubated with MTS in phosphate-buffered saline (pH 7.4) in the presence or absence ( )
of AsIII (ž), AsV (), MMAsV () or DMAsV (×) in vitro at a concentration ratio of arsenical : GSH = 5 : 1, and the absorbance
at 490 nm was measured during 120 min at 37 ◦ C. (B) Cys was
incubated with MTS in the presence or absence of various
concentrations of AsIII (ž), AsV (), MMAsV (), or DMAsV (×)
in vitro for 120 min at 37 ◦ C, and the absorbance at 490 nm
was measured. One representative experiment out of three similarly performed is given. Data are expressed as raw absorbance
(A) or metabolic integrity using the values from the control which
was incubated with only Cys and MTS as 100% (B). Results
are expressed as arithmetic mean plus/minus SEM (n = 3).
°
(lane 4). A putative DMAs–Cys conjugate spot (lanes 5–7;
Rf = 0.66) was detected after incubating 1 mM DMAsV with
≥3 mM Cys.
In our experimental conditions, AsIII was detected with
iodide vapor on HPTLC plate, but AsV could not be detected.
The spot density of AsIII separated on HPTLC plate was not
changed by the preincubation in distilled water for 48 h at
37 ◦ C before spotting; this means that AsIII was not oxidized
to AsV in aqueous solution during 48 h incubation (data not
shown).
Effect of the preincubation of MMAsV or
DMAsV with Cys on their cytolethality
Low (1–100 µM) or high (40 mM) concentrations of MMAsV
or DMAsV were preincubated with Cys at molar ratios of
MMAsV : Cys = 1 : 4 or DMAsV : Cys = 1 : 3 in distilled water
for 1 h at 37 ◦ C. TRL 1215 cells were exposed to these
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
Speciation Analysis and Environment
Effects of cysteine on arsenicals-induced cytolethality
(A)
1
2
3
4
5
6
7
1
2
3
4
5
6
7
(B)
Figure 3. MMAsV and DMAsV easily combine with Cys in
water. (A) MMAsV (1 mM) was incubated with (1, 4, 5 or 10 mM)
or without Cys in distilled water for 1 h at 37 ◦ C. After the
incubation, aliquots (25 µL) of these mixtures were spotted on
HPTLC plate, developed with solvent of ethyl acetate : acetic
acid : water (3 : 2 : 1), and the separated spots were detected
with iodide vapor. Lane 1, Cys (10 mM) only; lane 2, Cys–Cys
(10 mM) only; lane 3, MMAsV (1 mM) only; lane 4, MMAsV (1 mM)
plus Cys (1 mM); lane 5, MMAsV (1 mM) plus Cys (4 mM); lane
6, MMAsV (1 mM) plus Cys (5 mM); lane 7, MMAsV (1 mM) plus
Cys (10 mM). (B) DMAsV (1 mM) was incubated with (1, 3, 5 or
10 mM) or without Cys in distilled water for 1 h at 37 ◦ C. After
the incubation, aliquots (25 µL) of these mixtures were spotted
on HPTLC plate, developed with solvent of ethyl acetate : acetic
acid : water (3 : 2 : 1), and the separated spots were detected
with iodide vapor. Lane 1, Cys (10 mM) only; lane 2, Cys–Cys
(10 mM) only; lane 3, DMAsV (1 mM) only; lane 4, DMAsV (1 mM)
plus Cys (1 mM); lane 5, DMAsV (1 mM) plus Cys (3 mM); lane 6,
DMAsV (1 mM) plus Cys (5 mM); lane 7, DMAsV (1 mM) plus Cys
(10 mM).
arsenical–Cys mixtures at final arsenic concentrations up
to 100 µM for 48 h, and cellular viability was then assessed.
Copyright  2006 John Wiley & Sons, Ltd.
Figure 4. Effect of exogenous Cys on the cytolethality
of MMAsV or DMAsV ; the mixtures of high-concentration
MMAsV or DMAsV with Cys showed strong cytolethality. Low
(1–100 µM; ) or high (40 mM; ž) concentrations of MMAsV
(A) or DMAsV (B) were preincubated with Cys at molar ratios of
MMAsV : Cys = 1 : 4 or DMAsV : Cys = 1 : 3 in distilled water for
1 h at 37 ◦ C. After the incubation, TRL 1215 cells were exposed
to these arsenical–Cys mixtures at final arsenic concentrations
up to 100 µM for 48 h at 37 ◦ C. Cellular viability was then
assessed by the AlamarBlue assay. Results are expressed as
metabolic integrity using the values from control cells, which
were incubated with medium alone for 48 h at 37 ◦ C, as 100%.
Arithmetic mean plus/minus SEM of three separate experiments
performed in triplicate, n = 9. a p < 0.001, in comparison to
control cells.
°
As shown in Fig. 4, the mixture of low-concentrations of
MMAsV or DMAsV with Cys was not cytotoxic. However,
the mixture of high concentrations of MMAsV or DMAsV
with Cys showed strong cytotoxicity (LC50 values as As; the
mixture of 40 mM MMAsV with 160 mM Cys = 2.1 µM, the
mixture of 40 mM DMAsV with 120 mM Cys = 1.3 µM). The
same concentrations (up to 100 µM) of MMAsV or DMAsV
alone were not cytotoxic.
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
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T. Sakurai et al.
Speciation Analysis and Environment
DISCUSSION
Figure 5. Effect of exogenous Cys on the cytolethality of
the MMAs–Cys or DMAs–Cys mixtures. MMAsV or DMAsV at
40 mM was preincubated with 160 or 120 mM Cys, respectively,
in distilled water for 1 h at 37 ◦ C. TRL 1215 cells were exposed
to these MMAs–Cys mixture (A) or DMAs–Cys mixture (B) at
final arsenic concentrations up to 100 µM for 48 h at 37 ◦ C in the
presence ( ) or absence (ž) of 5 mM Cys. Cellular viability was
then assessed by the AlamarBlue assay. Results are expressed
as metabolic integrity using the values from control cells, which
were incubated with medium alone for 48 h at 37 ◦ C, as 100%;
arithmetic mean plus/minus SEM of three separate experiments
performed in triplicate, n = 9. a p < 0.001, in comparison to
cells exposed to the same concentrations of arsenicals in the
absence of Cys. b p < 0.01, c p < 0.05.
°
Effect of exogenous Cys on the cytolethality of
the MMAs–Cys or DMAs–Cys mixture
We also investigated the effects of exogenous Cys on the
cytolethality of the MMAs–Cys or DMAs–Cys mixture.
MMAsV or DMAsV at a high concentration, 40 mM, was
preincubated with 160 or 120 mM Cys, respectively, for 1 h
at 37 ◦ C. TRL 1215 cells were exposed to various arsenic
concentrations of these MMAs–Cys or DMAs–Cys mixtures
for 48 h in the presence or absence of 5 mM Cys. It was found
that exogenous Cys substantially reduced the cytolethality of
the MMAs–Cys or DMAs–Cys mixture (Fig. 5).
Copyright  2006 John Wiley & Sons, Ltd.
Arsenic intoxication occurs in many areas through the consumption of contaminated well water or foods containing
inorganic arsenicals.2 Inorganic AsIII has recently emerged
as an outstanding chemotherapeutic agent with remarkable
efficacy for certain human cancers such as acute promyelocytic leukemia.3,4 In many mammalian species, inorganic AsV
is first reduced to AsIII and is subsequently methylated to
MMAsV and DMAsV .5 – 7 The methylation of inorganic AsIII
and AsV was initially thought to be a detoxification process,8,9
however, it was recently reported that toxic MMAsIII and
DMAsIII might be produced through the methylation of inorganic arsenicals.10 – 12,25 – 27 Scott et al. reported that a thiol
reagent, reduced glutathione (GSH), reduced MMAsV and
DMAsV to MMAsIII and DMAsIII , resulting in the formation of monomethylarsonous diglutathione (MMAsIII DG)
and dimethylarsinous glutathione (DMAsIII G).28 We previously demonstrated that arsenicals and GSH concentrations
greatly affected the formation and cytolethality of trivalent
arsenicals.17 In the human body, other thiol reagent such as
Cys may also reduce pentavalent methylated arsenicals to
trivalent methylated arsenicals. Some studies have suggested
that Cys may enhance arsenic cytolethality;13 – 15 however, not
much is known concerning the effects of exogenous Cys on
the cytolethality of arsenicals. Thus, studying the effects of
Cys on the cytolethality of inorganic and organic arsenicals is
important in order to define their in vivo cytolethality.
Cellular GSH depletion with BSO increased the cytolethality of AsIII , AsV and MMAsV , and reversely decreased the
cytolethality of DMAsV . We previously demonstrated that
cellular GSH played an important role in the cytolethality of arsenicals; Cellular GSH prevented the cytolethality
of AsIII , AsV and MMAsV , but was required for DMAsV induced cytolethality.8,17 – 19,21 – 23 AsIII -induced cytolethality
was significantly decreased by the addition of Cys, however, this preventive effect of Cys disappeared when cellular GSH synthesis was stopped by BSO treatment. It has
been reported that the treatment with Cys increase cellular GSH concentration when cellular GSH concentration
is decreased, although Cys does not increase GSH concentration under normal, nonstressed, condition.29,30 It has
been reported that AsIII consumes the cellular GSH when it
shows cytolethality.8,17 – 19,21 – 23 These results suggested that
Cys decreased AsIII -induced cytolethality by increasing cellular GSH content. AsIII markedly enhanced the Cys-MTS
reaction, and we previously reported that AsIII also significantly enhanced the GSH-MTS reaction.18,19 It has still not
been clarified why AsIII enhances these reactions; however,
it is believed that this might, at least in part, be related to
the mechanism of AsIII -induced cytolethality or the protective
effects of Cys against AsIII -induced cytolethality. In contrast,
Cys did not affect AsV -induced cytolethality. The effect of
AsV on the reaction between Cys and MTS was very weak. In
the previous study, the effect of AsV on the reaction between
GSH and MTS was also found to be very weak.18 These results
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
Speciation Analysis and Environment
indicate that AsV might weakly react with thiol reagents such
as Cys or GSH. Further research is needed to verify the
unknown role of nonenzymatic chemical reactions between
inorganic arsenicals and thiol reagents on arsenical-induced
cytolethality.
The addition of ≥5 mM Cys increased MMAsV - and
DMAsV -induced cytolethality in cellular GSH-depleted cells.
In this study, we demonstrated that MMAsV and DMAsV
could easily combine with Cys in vitro using MTS assay
and HPTLC system. These results indicated that exogenous
Cys produced the cytotoxic arsenical–Cys conjugates. Scott
et al. reported that MMAsV or DMAsV combined with GSH
at molar ratios of MMAsV :GSH = 1 : 4 or DMAsV : GSH =
1 : 3 and formed MMAsIII DG or DMAsIII G in vitro.28 We
confirmed that the MMAs–Cys conjugate, which might be
monomethylarsonous dicysteine (MMAsIII DC), was formed
during the incubation of 1 mM MMAsV with ≥4 mM
Cys and that the DMAs–Cys conjugate, which might be
dimethylarsinous cysteine (DMAsIII C), was formed during
the incubation of 1 mM DMAsV with ≥3 mM Cys. These
results indicate that Cys reacts with MMAsV or DMAsV
at the molar ratios of MMAsV :Cys = 1 : 4 or DMAsV : Cys =
1 : 3 and forms cytotoxic MMAsIII DC or DMAsIII C in vitro.
When MMAsV or DMAsV was preincubated with Cys
at molar ratios of MMAsV :Cys = 1 : 4 or DMAsV : Cys =
1 : 3, MMAsV or DMAsV was not cytotoxic when mixed
in micromolar concentrations with Cys; however, when
mixed in millimolar concentrations with Cys, MMAsV or
DMAsV became strongly cytotoxic at the same final arsenic
concentrations. These results suggest that the formation
of cytotoxic MMAsIII DC and DMAsIII C depends on the
arsenicals and Cys concentrations, and millimolar levels of
both pentavalent methylated arsenicals and Cys are needed
to form the arsenical–Cys conjugates that show significant
cytolethality. It has been reported that arsenic concentrations
in the plasma of chronic arsenic poisoning patients is at
the micromolar level.31 Therefore, it is not probable that large
amounts of cytotoxic MMAsIII DC and DMAsIII C are produced
nonenzymatically at in vivo levels of arsenicals and Cys.
Cys alone at concentrations of 5 mM or above showed
significant cytolethality. Thus, the addition of very high
concentrations, 10 mM, of MMAsV or DMAsV and ≥5 mM Cys
might induce simple additive and/or synergistic cytotoxic
effects of Cys, methylated arsenicals, and methylated
arsenical–Cys conjugates.
On the contrary, the addition of <5 mM Cys with
MMAsV or DMAsV decreased MMAsV - and DMAsV -induced
cytolethality in the presence of cellular GSH. We confirmed
that MMAsV and DMAsV could directly react with Cys
in vitro. Additionally, exogenous Cys significantly decreased
the cytolethality of the MMAs–Cys mixture and DMAs–Cys
mixture. It was suggested that other synthetic trivalent
methylated arsenicals, such as MMAsIII O and DMAsIII I,
became MMAsIII (OH)2 and DMAsIII OH in aqueous solution
and showed significant cytolethality; their LC50 values
were micromolar range.32 – 34 We also recently demonstrated
Copyright  2006 John Wiley & Sons, Ltd.
Effects of cysteine on arsenicals-induced cytolethality
HO
4Cys
O
3H2O + Cys-Cys
Cys
AsIII Cys
H3C
Monomethylarsonous Dicysteine
(MMAsIIIDC)
AsV
OH
H3C
Monomethylarsonic Acid
(MMAsV)
Not Toxic
Not Toxic
2H2O + O2
?
HO
O2
AsIII
OH
2Cys-Cys
H3C
Monomethylarsonous Acid
(MMAsIII)
Very Toxic
H3C
3Cys
O
V
2H2O + Cys-Cys
H3C
III
As
Cys
H3C
Dimethylarsinous Cysteine
(DMAsIIIC)
As
OH
H3C
Dimethylarsinic Acid
(DMAsV)
Weakly Toxic
Not Toxic
2H2O + O2
?
O2
H3C
AsIII OH
H3C
Dimethylarsinous Acid
(DMAsIII)
Very Toxic
2Cys-Cys
Figure 6. The putative nonenzymatic chemical reactions of
monomethylarsenic or dimethylarsenic compounds with Cys.
that MMAsIII DG or DMAsIII G, which was produced by
incubation with MMAsV or DMAsV and GSH, might exert its
cytolethality by separating into MMAsIII or DMAsIII and
glutathione. These compounds are probably transformed
into MMAsIII (OH)2 or DMAsIII OH and are transported
into cells. Further, exogenous GSH maintains the form of
DMAsIII G and this conjugate cannot be transported into
cells because the GSH molecule is not transported efficiently
into cells.17 Cys did not significantly augment cellular
GSH concentration. It was reported that the Cys molecule
was not transported efficiently into the cells under normal
conditions.29,30 Thus, it would be indicated that exogenous
Cys less than 5 mM would partly produce the arsenical–Cys
conjugates in the cell culture medium, prevented the
cellular arsenic uptake, and then decreased MMAsV or DMAsV -induced cytolethality. When the arsenical–Cys
conjugates show significant cytolethality, they may separate
into trivalent methylated arsenicals and Cys, probably
becoming MMAsIII (OH)2 and DMAsIII OH, before being
transported into the cells (Fig. 6). Further examinations
are needed to determine the chemical characteristics of
the MMAs–Cys or DMAs–Cys conjugates in cell culture
Appl. Organometal. Chem. 2006; 20: 549–556
DOI: 10.1002/aoc
555
556
T. Sakurai et al.
medium.
Thiol reagents, such as Cys and GSH, may be key molecules
in preventing or inducing arsenic cytolethality. The present
results suggested that arsenicals and Cys concentrations
greatly affected the formation and cytolethality of these
arsenical–Cys conjugates. Arsenicals and Cys concentrations
higher than millimolar levels are needed to form the
arsenical–Cys conjugates with significant cytolethality. The
arsenical–Cys conjugates may exert their cytolethality by
separating into trivalent methylated arsenicals and Cys, and
being transported into the cells. In vivo levels (micromolar
levels) of arsenicals and Cys may not nonenzymatically
produce sufficient amounts of the cytotoxic arsenical–Cys
conjugates. Therefore, the significant cytolethalities of
MMAsIII DC and DMAsIII C may never manifest in the normal
human body. Further research will be required in order to
determine the role of Cys and methylation in the cytolethality
of arsenicals in chronic arsenic poisoning patients who
regularly ingest arsenic-contaminated well water and/or in
acute promyelocytic leukemia patients who are injected with
AsIII as a chemotherapeutic agent.
Acknowledgments
We express our thanks to Miss Satomi Murota, Miss Chihiro Kawata,
Mr Kouichiro Matsuda, Miss Tomoe Sakota and Mr Hiroki Soejima
(Tokushima Bunri University, Tokushima, Japan) for their excellent
technical assistance.
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DOI: 10.1002/aoc
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